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Carbon nanoparticles as possible

radioprotectors in biological systems

Article in Radiation Physics and Chemistry · July 2016

DOI: 10.1016/j.radphyschem.2016.07.006

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Carbon nanoparticles as possible radioprotectors in

biological systems

Anita Krokosz, Anna Lichota, Katarzyna E.

Nowak, Jacek Grebowski

www.elsevier.com/locate/radphyschem

PII: S0969-806X(16)30215-8
DOI: http://dx.doi.org/10.1016/j.radphyschem.2016.07.006
Reference: RPC7203
To appear in: Radiation Physics and Chemistry
Received date: 31 March 2016
Revised date: 2 July 2016
Accepted date: 5 July 2016
Cite this article as: Anita Krokosz, Anna Lichota, Katarzyna E. Nowak and Jacek
Grebowski, Carbon nanoparticles as possible radioprotectors in biological
s y s t e m s , Radiation Physics and Chemistry,
http://dx.doi.org/10.1016/j.radphyschem.2016.07.006
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 Carbon nanoparticles as possible radioprotectors in biological systems

Anita Krokosza*, Anna Lichotaa, Katarzyna E. Nowaka, Jacek Grebowskib

a
Department of Molecular Biophysics, Division of Radiobiology, Faculty of Biology and
Environmental Protection, University of Lodz, 141/143 Pomorska Street, 90-236 Lodz, Poland
b
Division of Technology, Faculty of Chemistry, University of Warsaw, Pasteura 1, 02-093
Warsaw, Poland

*
Corresponding author: krokosz@biol.uni.lodz.pl (Anita Krokosz)

Abstract

Ionizing radiation causes radiolysis of water and the production of reactive oxygen species
(ROS), which interact with biochemically important molecules in cells leading to cell death. In
order to reduce the dangerous radiation effects on cells, tissues and organs, the search for
radioprotectors is essential. ROS result in damage to biomolecules, e.g. proteins, lipids and
DNA, and as a consequence, cause the loss of cell function. The chemical and biological
properties of fullerenes and other carbon nanoparticles enable the possibility of generating either
oxidative stress or its attenuation by both scavenging free radicals and modification/upregulation
of endogenous antioxidative systems in cells. This study discusses the possible applications of
carbon nanoparticles as radioprotective agents and/or free radical scavengers. Special attention is
paid to water-soluble fullerenes as they are promising radioprotectors and exhibit low toxicity
and cytotoxicity.

Abbreviations: ADH, alcohol dehydrogenase; BSA, bovine serum albumin; C60HyFn, hydrated
C60 fullerene; CNTs, carbon nanotubes; DF-1, dendro[C60]fullerene-1; DRF, dose reducing
factor; DSBs, DNA double-strand breaks; DU145, prostatic adenocarcinoma cell line; ESR,
electron spin resonance; γH2AX, phosphorylated histone H2AX; GO, graphene oxide; GPx,
glutathione peroxidase; GSH, reduced glutathione; IC50, half maximal inhibitory concentration
or dose required to kill half of the cells; LDH, lactate dehydrogenase; LET, Linear Energy
Transfer; MDA, malondialdehyde; MWCNTs, multi-walled carbon nanotubes; NDs,
nanodiamonds; PBLs, peripheral blood lymphocytes; MiaPaCa2, pancreatic adenocarcinoma;
PBMCs, peripheral blood mononuclear cells; ROS, reactive oxygen species; RNS, reactive
nitrogen species; SOD, superoxide dismutase; SWCNTs, single-walled carbon nanotubes;
WBCs, white blood cells.

Keywords: carbon nanoparticles; fullerenes; fullerenols; free radicals; ionizing radiation;

biological systems

1. Introduction

Carbon-based nanoparticles (fullerenes and their derivatives, graphene, nanodiamonds and

carbon nanotubes) are a broad class of materials that have potential biomedical applications
(Melanko et al., 2009; Solarska-Ściuk et al., 2014; Vlasova et al., 2016; Wu et al., 2014). The
size, shape and structure of the nanoparticles allow for the creation of many new derivatives.
Unfortunately, all carbon nanoforms have a negative feature related to their tendency for
aggregation. They are often sparingly soluble or insoluble in polar solvents. Because of their
limited solubilization and tendency to aggregate, to study the reactivity of carbon nanoparticles
is rather difficult. One of the methods to reduce the aggregation of carbon nanoparticles is
functionalization with hydrophilic groups or conjugation with polar molecules/biomolecules
(Assemi et al., 2010; Vazquez et al., 2014).

Since its discovery in 1985, fullerene C60 (Fig. 1A) is one of the most investigated carbon
nanomaterials. In biomedical sciences, the water-soluble, hydrophilic derivatives of fullerene C60
are of great importance. Due to their unique structures with many π electrons, fullerenes undergo
characteristic reactions of additions to double bonds (Djordjevic et al., 2015; Grebowski et al.,
2014). However, fullerenes may produce reactive oxygen species (ROS) via photosensitization,
generating both singlet oxygen (1O2) and superoxide anion radicals (•O2−) (Pickering and
Wiesner, 2005; Wielgus et al., 2010).

Fullerenes can be modified to water-soluble forms by the addition of polar groups, e.g. hydroxyl
groups to produce fullerenols (Fig. 1B) or by conjugation with biomolecules, polymers or
dendrimers (Fig. 1C) (Afreen et al., 2015; Li et al., 2015).

Graphene is a single-atom thick, two-dimensional sheet of hexagonally arranged carbon atoms

with very interesting physical and chemical properties (Geim, 2009). Graphene oxide (GO) (Fig.
2B) is a graphene derivative that can be potentially applied in biomedicine for drug delivery,
tissue engineering and imaging (Shi et al., 2013; Wu et al., 2014; Yao et al., 2016).

Nanodiamonds (NDs) (Fig. 2A) are particles with a very narrow size distribution that averages 5
nm. X-ray and neutron diffraction measurements and transmission electron microscopy analysis

2
have confirmed that the core of a ND is an sp3-bonded diamond lattice. NDs have proved useful
for targeted drug delivery, generating free radicals for therapy of solid tumors, immobilizing
proteins for purification and for cell imaging (Holt, 2007; Passeri et al., 2015; Petit et al., 2014).

Carbon nanotubes (CNTs) (Fig. 3) could be used in drug delivery, bio-analysis and diagnostics
because of their high electrical conductivity and strength (Khanna et al., 2015; Yang et al.,
2010). Recently, CNTs have appeared as a promising scaffold material for in vitro culture of
cells for their possible use in tissue engineering and regenerative medicine (Dinicola et al., 2015;
Hopley et al., 2014).

Ionizing radiation acts on living organisms and their tissues and cells, mainly by products of
water radiolysis. Under aerobic conditions, the products of water radiolysis are ROS e.g.
hydroxyl radicals (•OH), superoxide anion radicals (•O2−) and hydrogen peroxide (H2O2)
(Rodacka, 2016). In addition to ROS, reactive nitrogen species (RNS) can also be generated in
cells. Ionizing radiation generates the same free radicals as conditions of other oxidative stresses
(Bors et al., 2011; Houée-Levin and Bobrowski, 2013; Marczak and Bukowska, 2013;
Sosnowska et al., 2015).

Exposure to ionizing radiation, for example during radiotherapy and accidental exposures, causes
damage to normal tissues and total body radiation exposures can result in lethality (Azzam et al.,
2016; Painuli and Kumar, 2016). In order to reduce dangerous radiation effects on healthy
tissues and organs, radioprotective agents are being searched for. It was demonstrated in
numerous works, reviewed by Gudkov et al., 2015, that some compounds could play a protective
role against the effects of excessive exposure of biological systems to ionizing radiation.

Radioprotective agents are used in the preventive treatment of individuals involved in dealing
with the consequences of accidental or terrorist exposures, as well as for patients who are
undergoing radiation therapy (Gudkov et al., 2015). According to Gudkov et al. (2015), an ideal
radioprotector must meet the following requirements: high efficiency, lack of toxicity,
convenient administration pathway, low manufacturing costs, storage stability, long duration of
protective effect and the ability to alleviate effects of various types of ionizing radiation.
Selective radioprotection, i.e. protection of non-tumor tissue with relevant low protection of
cancer tissue, is essential for patients undergoing radiotherapy. There are several ways to achieve
selective radioprotection, one is based on the difference in metabolism of non-tumor and tumor
cells, another on the difference in signaling pathways (cascades) and a further one on the

3
difference in the transport of radioprotective agents to tissues (Brown et al., 2010; Painuli and
Kumar, 2016).

Both ROS and RNS result in damage to biomolecules, e.g. proteins, lipids and DNA, and as a
consequence cause the loss of cell function. The chemical and biological properties of fullerenes
and other carbon nanoparticles enable the possibility of either generating oxidative stress or its
attenuation by both scavenging free radicals and modification/upregulation of endogenous
antioxidative systems in cells.

In this study, the possible applications of carbon nanoparticles as radioprotective agents and/or
free radical scavengers are discussed. Special attention is paid to water-soluble fullerenes as they
are promising radioprotectors and exhibit low systemic toxicity and cytotoxicity.

2. Radioprotective effects of fullerene C60 derivatives

2.1. Fullerene C60 derivatives

There are numerous reports on the antioxidant properties of fullerene C60 (Fig. 1A) derivatives
and their protective effect in oxidative stress conditions (Cai et al., 2008; Ehrich et al., 2011;
Grebowski et al., 2014; Saitoh et al. 2011; Yin et al 2009). However, there are also reports that
fullerenes may have cytotoxic properties (Johnson-Lyles et al., 2010; Wielgus et al., 2010). The
factors that may condition the cytotoxicity are the complex structure of the nanoparticles and
their ability to scavenge or generate ROS, differences in size and shape of the molecules,
production method and the presence of contaminants.

2.2. Water-soluble derivatives of fullerene C60

The attachment of hydrophilic functional groups (e.g. OH) on the surface of a C60 molecule (Fig.
1A) increases its solubility in water and thus its usage in biological systems or biomedical
applications also increases (Djordjevic et al., 2015; Partha and Conyers, 2009). Water-soluble
derivatives of fullerene C60, due to their ability to scavenge free radicals and ROS, as well as
anti-oxidative effects (Andrievsky et al., 2009; Vavrova et al., 2010), were extensively tested for
protective properties against ionizing radiation in different model systems. The radioprotective
effects of fullerenols and other water-soluble derivatives of fullerene in various chemical and
biological systems are summarized in Tables 1 and 2, respectively.

4
2.2.1. Fullerenols C60(OH)n

The effects of fullerenols, C60(OH)n (n > 18) (Fig. 1B), on different systems irradiated with low-
LET ionizing radiation (i.e. X- and -rays) have been studied in the past decade (Table 1). The
first literature reports on radioprotective properties of fullerenols appeared in 2005 (Trajkovic et
al., 2005; Zhao et al., 2005). Zhao et al. (2005) reported the protective impact of C60(OH)18-22 (in
a concentration range of 0.06–0.1 mg/mL) on protozoan S. mitylus exposed to gamma
irradiation. It was also shown that at doses greater than 0.25 mg/mL, fullerenol exhibited
cytotoxic actions, as evidenced by a reduction survival below the control value. The group of
Trajkovic (2005, 2007) reported that C60(OH)24 caused a protective effect that increased the
survival of X-irradiated experimental mice and rats. These authors (2007) also compared
radioprotection by C60(OH)24 with that of amifostine. It was noted that fullerenol causes similar
effects at lower concentrations (100 mg/kg) than amifostine (300 mg/kg). Bogdanovic et al.
(2008) showed that pretreatment with C60(OH)24 at a concentration of 10 μM protects K652 line
cells against 24 Gy of -rays, as evidenced by an increase in the antioxidative enzyme (SOD and
GPx) activity. The results of studies carried out by Cai et al. (2010) indicate that C60(OH)24
pretreatment effectively increases the survival of whole body irradiated mice without apparent
toxicity, and decreases oxidative damage while enhancing immune and mitochondrial functions.

Recently, the effect of highly polyhydroxylated fullerene derivatives (n > 30) on X-irradiated
enzyme ADH (Krokosz et al., 2014) and human PBMCs (Nowak et al., 2014) was evaluated
(Table 1). It was shown that C60(OH)~30 in a concentration of 75 mg/L protected ADH against
radiation-induced inactivation and did not affect its activity (Krokosz et al., 2014). In turn,
Nowak et al. (2014) reported that after pretreatment with C60(OH)36 (75 and 150 mg/L), an
increase of viability of PBMCs exposed to high doses of X-rays and 48 h of post-irradiation
incubation was observed, but there was no impact of C60(OH)36 on DNA damages. Fullerenol at
both concentrations was not cytotoxic to PBMCs after 24 and 48 h of incubation. The authors
suggested that interactions of C60(OH)36 with the surface of plasma membrane, not inside the
cells, are responsible for the observed effect (Nowak et al., 2014).

2.2.2. Other water-soluble derivatives of fullerene C60

Since 2006, a water-soluble derivative of fullerene (dendro[C(60)]fullerene; DF-1) (Fig. 1C) has
been extensively evaluated as a radioprotector (Brown et al., 2010; Daroczi et al., 2006; Theriot
et al., 2010) (Table 2). First, it was reported that DF-1 protected zebrafish against irradiation

5
with γ-rays (Daroczi et al. 2006). In turn, Brown et al. (2010) reported that DF-1 has modest
radioprotection of both human fibroblast (MRC5) and cancer (DU145 and MiaPaCa2) cell lines,
as well as experimental mice. Also, there was no selective radioprotection for normal and tumor
cells (Brown et al., 2010). The effect of DF-1 on irradiated (up to 4 Gy of γ-rays) human
peripheral blood lymphocytes (PBLs) and rat crypt line cells (IEC-6) was studied by Theriot et
al. (2010). The authors reported that DF-1 had a radioprotective effect against oxidative stress,
DNA damage and the death of the studied cells.

Hydrated C60 fullerene (C60HyFn) was studied for antiradical activity and radioprotective
efficiency (Table 2) by Andrievsky et al. (2009). It was reported that C60HyFn showed apparent
antiradical activity in the concentration range of 10−11–10−6 M (DRF 1.1 for 10−11 and 14.87 for
10−6 M). The radioprotective effect of C60HyFn has been demonstrated in concentrations of 10−7–
10−6 M on DNA (DRF 1.58 and 1.97 for 10−7 and 10−6 M, respectively) and in a dose of 1 mg/kg
b.w. on mice (Andrievsky et al., 2009).

Table 1. Radioprotective effects of fullerenols in different experimental systems.

Type of
Model
Fullerenol radiation/dose Main findings/effects References
system
[Gy]
γ-rays ˗ protection from damage induced by radiation
Stylonychia Zhao et al.,
C60(OH)18-22 (60Co)/100– ˗ radioprotection depends on both fullerenol
mytilus 2005
2000 concentration and radiation dose
˗ dose-dependent increase of survival of Trajkovic et
mice X-rays/6–8
irradiated animals (10 and 100 mg/kg) al., 2005
˗ protection against radiation-induced mortality
˗ better protection at a dose of 100 mg/kg than
at a dose of 10 mg/kg
˗ more pronounced radioprotective effects on
Trajkovic et
Wister rats X-rays/7, 8 the spleen, small intestine and lung, but less
al., 2007
radioprotective effects in protection of the
heart, liver and kidney
˗ prevention radiation-induced reduction in the
C60(OH)24
WBC count
human
erythromyel
oblastoid Bogdanovic
X-rays /24 ˗ upregulation of the activities of SOD and GPx
leukemia et al., 2008
cell line
(K562)
˗ reduction of irradiation-induced mortality
60 Cai et al.,
mice γ-rays ( Co)/8 after 2 week pretreatment with fullerenol
2010
without apparent toxicity

6
 ˗ protection of the irradiation-induced reduction
of WBC counts
˗ decrease of oxidative damages: inhibition of
protein carbonyl formation and lipid oxidation
(MDA level), increased SOD activity and
GSH levels
˗ protection against the ionizing-radiation-
induced con A-induced decrease in cell
proliferation
˗ prevention of the ionizing-radiation-induced
increase in the number of splenocytes (from
irradiated animals) in sub-G1 phase
˗ no effect on the unirradiated enzyme activity
˗ protection against radiation inactivation due
to simple competition for the •OH radicals
Krokosz et
C60(OH)~30 ADH X-rays/0–100 ˗ reduction of the ability to prevent the
al., 2014
formation of protein peroxides
˗ decrease in the radiation yield of inactivation
of enzymes irradiated with fullerenol by 20%
human ˗ increase in the viability of X-irradiated cells
peripheral after 48 h of post-irradiation incubation Nowak et
C60(OH)36 X-rays/0–50
blood ˗ increase in lymphocyte granularity without al., 2014
lymphocytes causing cytotoxicity

Table 2. Radioprotective effects of water-soluble derivatives of fullerene C60 in different

experimental systems.

Type of
Fullerene Model
radiation/dose Main findings/effects References
derivative system
[Gy]
˗ time-dependent radioprotection (before or
zebrafish γ-rays (137Cs) Daroczi et
immediately after irradiation DF-1 treatment
Danio rerio /20–80 al., 2006
(100 µM))
human ˗ moderate toxicity (100 µM) after 24 h
peripheral incubation
blood ˗ no radioprotection after 24 h post-irradiation
γ-rays (137Cs)/0– Theriot et
lymphocytes ˗ radioprotection after 48h post-irradiation
DF-1 4 al., 2010
˗ protection against radiation-induced DNA
rat crypt cell
damage (micronuclei test)
line (IEC-6)
˗ reduction of ROS level in cells
human lung ˗ no effect on cellular radiosensitivity (10 μM)
fibroblast Brown et al.,
X-rays/0–6
cell line ˗ dose reducing factor (DRF) 1,1 (for 100 μM) 2010
(MRC5)

7
 ˗ no effect on induction of DSBs (γH2AX foci)
human
(10 and 100 μM)
prostatic
˗ inhibition of DNA damage after 1 and 6 h
carcinoma
after radiation (only 100 μM)
cell line
˗ no protection after 24 h from irradiation (10
(DU145)
X-rays/0–8 and 100 μM)

human
pancreatic
carcinoma ˗ no protection (100 μM)
cell line
(MiaPaCa2)
γ-rays (137Cs)/6– ˗ increase in the 30 day survival of mice treated
mice
11 with total body irradiation (300 mg/kg)
double
˗ apparent antiradical activity in the range of
distilled
concentrations of 10−11–10−6 M
water X-rays/1–7
˗ protection of DNA against radical-induced
DNA Andrievsky
C60HyFn damage (in concentrations of 10−7–10−6 M)
et al., 2009
˗ 15% survival rate of irradiated animals after
30 days
mice X-rays/7
˗ prevention of weight loss characteristics for
radiation impact

Summarizing the data presented in Tables 1 and 2, water-soluble derivatives of fullerene C60
(e.g. fullerenols C60(OH)n, dendrofullerene and hydrated C60 fullerene) have antioxidant
properties and can reduce damage in irradiated biomolecules and cells or increase the survival of
irradiated organisms. Moreover, these substances are low or moderate in toxicity in the studied
systems (Tables 1 and 2). In view of these properties, water-soluble derivatives of fullerene are
interesting for researchers searching for potential radioprotectors. It is known that basic
conditions must be met by substances for the protection of healthy tissues from ionizing
radiation effects, including selective protection of normal tissues and a lack (or minimum) of
toxicity for the organism (Gudkov et al., 2015; Vavrova et al., 2012).

Generally, to study radioprotective effects, water-soluble derivatives of fullerene in non-toxic

doses were used and ionizing radiation in doses graded for biomacromolecules/cells or lethal for
organisms were applied (Tables 1 and 2). Taking into account the fact that in the literature there
are reports on the cytotoxicity of these compounds, C60(OH)24 is cytotoxic in kidney cells (LLC-
PK1) (Johnson-Lyles et al., 2010) and human retinal pigment epithelial line cells (Wielgus et al.,

8
2010), the short and long term side effects on normal tissues resulting from the usage of water-
soluble fullerenes should also be considered.

3. GO, NDs and CNTs as oxidative stress attenuators or enhancers

There is limited data on the possible radioprotective properties of GO (Fig. 2B), NDs (Fig. 2A)
and CNTs (Fig. 3). These nanoforms of carbon are used preferably in materials engineering and
targeted drug therapy (Arya et al., 2013; Gupta et al., 2016; Przybytniak et al., 2016). However,
quite a number of studies have demonstrated that the mechanisms of action of carbon
nanoparticles involve ROS and carbon-centered free radicals (Moller et al., 2014). The
prooxidative or antioxidative properties of these carbon nanomaterials have been proven (Moller
et al., 2014; Nymark et al., 2014; Qiao et al., 2014).

Combustion-generated graphenic nanoparticles scavenge •OH depending upon the degree of

graphitization of the material and the structure of the particles (Carella et al., 2013). The addition
of •OH to carbon nanoparticles results in the introduction of functional groups such as hydroxyl
(-OH), carboxyl (-COOH) or carbonyl species (C=O) to the surface of the nanostructure. Free
radicals are scavenged to specific patches of the graphenic nanostructure resulting in the
insertion of acidic groups at the surface. Graphenic nanoparticles did not induce any toxic effect
in alveolar macrophages (MH-S cells), as measured by LDH release tests and oxidation of GSH
(Carella et al., 2013).

Arya et al. (2013) investigated the toxicity of SWCNTs (Fig. 3A) and GO (Fig. 2B) in
combination with paclitaxel against lung cancer cells (A549 and NCI-H460 lines). Both
nanoparticles demonstrated a concentration (50–150 mg/L) and time dependence increase in
toxicity via ROS generation. Combined treatment with SWCNTs or GO and paclitaxel led to a
significant enhancement in drug anticancer activity. The observed synergism could be attributed
to ROS generated by carbon nanoparticles. Enhancement in ROS production induced MAPK
activation which was demonstrated by enhanced BrdU incorporation. In addition, the
combination treatment resulted in cell death predominantly via apoptosis which is very desirable
in cancer therapy (Arya et al., 2013).

GO at a concentration of up to 10 mg/L did not exhibit cyto- and genotoxicity (Qiao et al., 2014).
Pretreatment of fibroblasts with 10 mg/L of GO markedly decreased X-ray induced damage to
DNA and protected fibroblasts against apoptotic death. The level of DNA damage and cell death

9
was reduced by 48% and 39%, respectively. Efficiency of GO in protection against oxidative
damage was only slightly weaker than that of melatonin, while CNTs at 10 mg/L did not entirely
protect fibroblasts against X-ray induced damage. CNTs (Fig. 3) cannot remove ROS generated
by X-rays as efficiently as GO (Fig. 2B) due to its different close structure. Nevertheless, GO at
higher concentrations induced severe DNA damage and cell death. At a concentration of 500
mg/L, the effect of GO was comparable to 2.5 Gy of X-radiation (Qiao et al., 2014). Thus, GO at
relatively low concentrations can be used as an effective radioprotective agent in therapeutic
applications.

CNT toxicity depends on the CNT type, the method of its synthesis, i.e. the presence of
impurities (especially transition metal impurities), its functionalization and cell line testing
(Coccini et al., 2010; Cui et al., 2010; Dinicola et al., 2015). Significant research has proven a
pronounced toxicity and cytotoxicity of CNTs. It is considered that CNTs are toxic because of
their fibrous-like morphology, which is comparable to that of asbestos (Fig. 3). However,
existing toxicity data are ambiguous (Nymark et al., 2014).

Free radical formation and the scavenging ability of MWCNTs (Fig. 3B) in cell-free and cellular
systems using human bronchial epithelial cells (BEAS 2B) were investigated by Nymark et al.
(2014). The results obtained indicate that MWCNTs may either produce or scavenge ROS. The
specific ROS formation linked to primary particle and aggregate size affects the cytotoxicity of
MWCNTs. It was shown that MWCNTs which are larger and open-structured aggregates exhibit
stronger cytotoxicity and faster sedimentation rates. MWCNTs were also found to scavenge
H2O2-generated •OH, but it was solution dependent (cell culture medium, buffer and the presence
of BSA). Needle-like MWCNTs (average diameter >74 and 64.2 nm, average length 5.7 and 4.0
μm, respectively) induced, in addition to a scavenging effect, a dose-dependent formation of a
unique, unidentified radical. The formation of •O2− by MWCNTs was also observed in cell-free
and BSA-free buffers (Nymark et al., 2014). Direct •O2− production by SWCNTs has previously
been detected by ESR spectrometry in normal and malignant human mesothelial cells (Pacurari
et al., 2008). SWCNTs have oxidative potential and generated oxidative stress in aortic
endothelial cells by increasing heme oxygenase-1 (HO-1) expression levels (Cheng et al., 2012).
Shvedova et al. (2014) proved the formation of carbon-centered lipid-derived radicals in the
heart and liver of mice exposed to SWCNTs by ESR spectrometry. However, a few studies have
indicated that some MWCNTs are efficient scavengers of •OH and •O2− radicals in cell-free
conditions. The scavenging of free radicals by CNTs was suggested to be related to the amount
and nature of defects in the CNTs, i.e. breaks in the graphene framework (Fenoglio et al., 2008).

10
NDs with a diameter of about 5 nm are the most useful for biomedical applications. The most
common method of ND synthesis is a detonation method based on the detonation of carbon-
containing explosives due to its low cost and simplicity. In addition, the possibility of
functionalizing the surface of NDs expands the scope of their applications, e.g. as drug
nanocarriers, antibacterial agents, therapeutic protein carriers or in imaging (Passeri et al., 2015).

NDs are also proposed to generate ROS which may be responsible for their toxicity in higher
concentrations. They can also be employed in cancer therapy. Petit et al. (2014) patented an
invention based on generating free radicals by NDs exposed to radiation, e.g. ionizing radiation
in solid tumors. In order to increase the effectiveness of the NDs, they can be complexed with a
radiosensitizer (such as a chemical molecule or an interfering RNA targeting a repairing gene).

NDs at low concentrations are non-cytotoxic or cytostatic to human peripheral lymphocytes

(IC50 = 50 mg/L), which was revealed by trypan blue exclusion and nuclear division index tests,
respectively. However, at lower concentrations NDs promoted genotoxicity and aneugenic
effects. NDs induced DNA single-strand breaks at a 1 mg/L concentration and oxidative DNA
damage and micronuclei formation at a concentration of 10 g/L. ND genotoxicity is surface
chemistry-specific since oxidized NDs induced more potent DNA damage than raw NDs. Total
ROS and specific superoxide production were concentration-dependent which confirms that the
ND-induced DNA damage was mediated by oxidative stress (Dworak et al., 2014).

4. Conclusions

One can see that among carbon nanoparticles, fullerenols and other water-soluble derivatives of
fullerene C60 are the most promising for protection against oxidative stress generated by low-
LET ionizing radiation in diverse model systems. Due to their unique physico-chemical
properties and benign toxicity profiles, fullerenols can be used at high enough concentrations to
scavenge free radicals and/or upregulate antioxidative systems in cells. However, a significant
number of studies have proven ROS-mediated toxicity of carbon nanoparticles in biological
systems. In particular, CNTs are toxic because of their fibrous-like morphology. However, the
results on triggering the formation of free radical toxicity by a variety of carbon nanoparticles are
inconsistent. ROS-mediated toxicity of carbon nanoparticles is closely connected with the
technological impurities present in the soot sample.

Acknowledgments

This work was supported by a statutory grant from the University of Lodz.

11
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Figure captions:

Fig. 1. Fullerene C60 (A), fullerenol C60(OH)n (B) and dendro[C60]fullerene (C).

Fig. 2. Nanodiamond (A) and graphene oxide (B). Adapted from Jin et al., 2015.

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Fig. 3. Single-walled carbon nanotube (SWCNT) (A) and multi-walled carbon nanotube
(MWCNT) (B). This research was originally published in JNM. (Reilly, 2007) © by the Society
of Nuclear Medicine and Molecular Imaging, Inc.

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 Highlights:

 Carbon nanoparticles can either generate or scavenge free radicals

 Water-soluble fullerenes are promising radioprotectors in biological systems
 GO, NDs and CNTs have limited radioprotective properties

20

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